Environmentally safe agricultural supplement

ABSTRACT

A substantially microbe-free composition containing plant growth regulators is produced by culturing a fungal spawn on high carbohydrate medium under suitable conditions. The culture filtrate is sterilized, formulated, and used to enhance plant growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) to U.S.provisional application Ser. No. 60/396,833, filed 16 Jul. 2002, thecontents of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to a formulation which enhances growth and qualityof food and ornamental crops. More specifically, the invention concernsa sterilized filtrate derived from composting high-sugar-content medium,preferably agricultural waste.

BACKGROUND ART

A wide variety of strategies has been used to enhance the productivityof food crops, including the use of fertilizers as nutritionalsupplements, application of pesticides to counteract the negative effectof infestation, and supplementation with growth hormones such as auxinsand gibberellic acids. Each of these approaches, especially whenimplemented using synthetic materials, poses problems with respect tounacceptable alteration of the environment and concomitant unbalancingof the ecosystem.

It is understood that plant growth regulators are produced by somefungi. For example, gibberellins, indole-acetic acid, cytokinins, andother compounds useful in regulating plant growth are found inBasidiomycetes as reviewed by Brizuela, M. A., et al., Revista IberoAmericana de Mycologgia (1998) 15:69-74; the plant growth regulatordihydroampullicin is produced by a fungus Ampulliferina (Kimura, Y., etal., Bioscience Biotech. & Biochem. (1993) 57:687-688) and it isgenerally known that Neurospora and various phytopathogenic fungiproduce plant growth regulators. It is also known that Polyporusversicolor, a white rot polypore produces plant growth regulators.However, culture conditions to enhance the production of plant growthfactors vary widely.

Basidiomycetes have been shown to produce gibberellins, auxins,indoleacetic acid, abscisic acid, cytokinins and ethylene, as well asother plant stimulatory metabolites. The production of these factors,however, has been shown in the context either of production when inassociation with a plant per se, or on small-scale laboratory bases.

The present invention provides a method to provide plant growth factorsfrom fungi, in particular Basidiomycetes, on a commercial scale. It hasbeen generally considered not possible to do this. For example,according to “Handbook of Applied Mycology,” Vol. 4, FungalBiotechnology (1992) page 588

-   -   It is interesting to note that the species of Taphrina and        Exobasidium formed yeasty like cells and spores in surface        layers on the parasitized tissues of the host plant. When grown        on submerged fermentation (SMF) process in synthetic media, the        cytokinin (CK) production by these fungi was too small to cause        extensive morphological changes that occur naturally in the host        plant in spite of the growth in SMF medium in the form of yeast        like cells. It was therefore stressed that the production of CK        by pathogenic hyphal cells of Taphrina growing on host tissues        was undoubtedly different in quantities. The close resemblance        of the growth of the fungi under solid state fermentation        technique to the above type of growth of the fungi on the host        tissue is well known.

Further, according to this source,

-   -   It is obvious that the obligate parasitic, mycorrhizal,        ascomycetous, and basidiomyceteous fungi have no potential in        fermentative production of CK (cytokinin) due to problems in        cultivation and slow growth rate.    -   Downstream processing involves handling of large volumes of        liquid for separating extremely low quantities of GA3        (Gibberellic Acid 3), and thus is cost intensive when compared        to other fermentation products such as citric or gluconic acids.    -   After separation of mycelial cells by filtration or        centrifugation, GA3 is either adsorbed on suitable        resins/adsorbents or extracted in appropriate solvents. Further        purification involves a series of operations such as repeated        liquid-liquid partitioning, concentration under vacuum, and        final processing to obtain amorphorus powder or crystals of GA3.

Thus, even if it is known that certain plant parasitic fungi may produceplant growth regulators, the fungi may not produce the plant growthregulators (PGR) in quantity in fermentation systems to be of practicaluse unless culturing, extraction and concentration steps are taken.

It has now been found that an environmentally friendly stimulant ofgrowth can be supplied by composting agricultural waste in the presenceof fungal spawn, sterilizing the culture filtrate, and applying theresulting “liquid compost factor (LCF)” directly to field crops evenafter diluting with water 1 to 500 or up to 1:10,000. There is no needfor extraction or chemical separation steps in order to obtain a useablesolution. Only heating and filtration are needed. No solvents or resinbeds are needed for extraction or concentration. The solids and filtermaterial itself from filtration step of the heated liquid culture fluidmay be dried and used as a source of plant growth stimulants as well.The dried material may be added to compost as an additive.

DISCLOSURE OF THE INVENTION

The invention is directed to a composition for stimulation of rapid rootgrowth and plant bulk, as well as enhanced productivity in field crops,trees and other plants, which composition comprises the pasteurizedculture filtrate of a fungal spawn grown, preferably on agriculturalwaste, but in any event, on medium with a high available carbohydrateconcentration. The resulting “Liquid Compost Factor (LCF)” can beapplied in a variety of ways to many crops to result in increased ratesof growth and yields.

Thus, in one aspect, the invention is directed to a method to prepare aplant supplement, which method comprises culturing fungal spawn on amedium with at least 10% available carbohydrate concentration in thepresence of light, and aeration under conditions whereby the mycelialmat is undisturbed, harvesting the culture, denaturing protein, removingsolids, and pasteurizing or otherwise sterilizing the filtrate, toobtain said composition. In another aspect, the invention is directed tothe composition prepared by this method. The invention composition thuscomprises the sterilized culture filtrate of a fungal spawn culturegrown in high carbohydrate medium, preferably comprising agriculturalwaste. In still another aspect, the invention is directed to methods toenhance plant growth and productivity using this composition, eitheralone or in combination with other growth promoters. Thus, the inventionis also directed to methods to culture plants using the compositions ofthe invention whether the invention compositions are used alone or incombination with, for example, herbicides, insecticides, nematicides, orother growth stimulators or nutrients.

MODES OF CARRYING OUT THE INVENTION

The invention is directed to an environmentally friendly plant growthstimulating composition designated “Liquid Compost Factor (LCF).” LCF isthe sterilized culture filtrate of a fungal spawn culture which has beengrown under specified conditions and in a medium high in carbohydrate.The compositions of the invention include the sterilized filtrate fromthe fungal spawn as well as dried forms thereof and of recovered solidsfrom filtration. Typically, either of these compositions is diluted inaqueous solution. The resulting diluted LCF, when applied to field cropssuch as corn, taro, lettuce, soy bean, cucumber, tomato, pineapple andother food crops, is able to stimulate root growth, enhance fruitingproductivity, and improve crop yield. LCF has been shown to alleviatenematode infestation symptoms in established crops, and aids in diseasereduction such as the reduction of fungal leaf spots, leaf rot andbacterial bulb rot. LCF may also be used on ornamental plants and isable to enhance flowering. It is effective on turf wherein it enhancesgreening. LCF can also be used to extend the life of cut flowers andfoliage. LCF is also useful in enhancing tree growth thus, for example,assisting reforestation efforts.

The LCF of the invention contains plant growth regulators which aresecondary metabolites of the fungal culture, as well as elicitors ofplant defense mechanisms. The production of these plant growthregulators is effected by appropriate culturing conditions, byappropriate medium corn position, and by proper post-culture treatment.The medium must contain sufficient available carbohydrate and sufficientpotassium ion to effect this production with the appropriate precursorsfor metabolic production of the PGR'S. Molasses contains about 2%-6%potassium and is a preferred, environmentally acceptable source,although other sources such as bananas, potatoes, prunes, oranges,tomatoes, artichokes, squash, grapes, sunflower, spinach, seeds oralmonds could be used as well. The final concentration of K⁺ should be0.005% to 0.1% wt/vol., preferably 0.01%-0.1% wt/vol.

In general, the higher the available carbohydrate content, the moreefficient the production of plant growth regulators; however, too high aconcentration of carbohydrate in the form of sugars would unacceptablyincrease osmotic pressure and thus retard or eliminate growth of thefungus. Other factors which enhance the production of plant growthregulators in the ultimate product include growth under conditions ofaeration under conditions wherein the mycelial mat is left undisturbedand in the presence of light predominantly in the long wavelengthportion of the visible spectrum. The lighting conditions suitable forculture are preferably those derived from U.S. Pat. No. 5,123,203, thecontents of which are incorporated herein by reference. By addition ofcarotenoid pigments and reduction of Ca⁺² in the fruiting substrates,red light sources were found to be preferred. Plant growth regulator(PGR) production is thus also enhanced by addition of carotene to themedium sufficient to produce a yellow color. If pineapple juice isemployed as a carbohydrate source, sufficient carotene is inherentlypresent.

The culture medium will contain an available sugar concentrationcorresponding to a content of 5-10% molasses. Although agriculturalwaste may be used to compose the medium, any source of suitablecarbohydrates and other required nutrients, including carotene, could beused; some portion of the nutrients may be supplied by the fungal spawnitself which is prepared by culturing fungi in the presence of grainsand other nutrients. Thus, the required sugar content of the medium canbe supplied by syrups prepared from any source, including variousfruits, corn syrup, sugar cane syrup, sugar beet syrup, molasses, andthe like. Syrups prepared from other fruits, such as pineapple, orange,plum, grape, papaya and many other may also be used. It is preferred touse plant extracts as a source of nutrients in the medium.

The medium must have an available carbohydrate content which is higherthan that typical for culturing of fungi. By “available carbohydrate” ismeant carbohydrate energy sources which are metabolizeable by theflingal culture. Typical components of these available carbohydrateinclude sucrose, glucose, other simple sugars and disaccharides.Typically, the medium will contain at least 10% wt/vol availablecarbohydrate, preferably 12% wt/vol, more preferably 13% wtlvol, andeven more preferably 15% wt/vol. Alternatively, the final concentrationin the medium results in a IIBRWII BRIX reading of at least 10, morepreferably at least 12, most preferably at least 15. High concentrationsof available carbohydrate are highly preferred and, as stated above, arelimited only by the necessity to avoid generating unacceptable osmoticpressure conditions. Since fungi are able to digest cellulose, enhancingthe carbohydrate levels in the form of cellulose, or other carbohydratewhich does not enhance osmotic pressure, may preferably be used.

It appears that optimal BRIX values for the culture medium are in therange of 12-15. In one typical culture, BRIX values above 19, e.g., 24or 30, resulted in either very slow growth or no growth at all. At 19BRIX, the mycelial covered the surface of the medium but in only a thinlayer; at 11 BRIX and 8 BRIX a very good growth is achieved. However, atbelow 11 BRIX, the PGR content appeared to be less.

In addition to the available carbohydrate as a carbon source, the mediummust also contain other nutrients, notably a source of nitrogen andvarious cofactors. Typically, there is sufficient source of most ofthese nutrients in the fungal spawn used for an inoculum. However, itappears important that the medium contain a concentration of carotenewhich is sufficient to provide a visible yellow color.

If molasses is used as at least a portion of the source for availablecarbohydrate, the molasses itself supplies many vitamins and othernutrients required by the fungus. Syrups prepared from sugar cane arepreferred to those prepared from sugar beet as these syrups provide abetter source of nutrients. Other sources of desirable nutrients includethe use of bananas for supply of potassium ion and papaya is also ahelpful addition to the medium. Papaya contains carotenoids, sugars, andsulfur compounds. It is particularly high in fructose.

The medium is first sterilized, preferably by heating to a sufficienttemperature for a sufficient time to remove any contaminating organisms.The decontaminated medium is then inoculated with a culture of fungus,i.e., a fungal spawn.

Any fungus can be used in the invention provided it is adaptable to theculture techniques described herein. While a multiplicity of fungi havebeen described as able to produce plant growth regulators, typically,this has not been the case as a means for commercial or practicalproduction of these compounds.

The culturing of fungi useable to obtain the LCF compositions of theinvention can be conducted in an efficient manner using readilyavailable equipment. While stainless steel drums are useful, they areexpensive and unless the stainless steel is especially formulated toresist corrosion, corrosion may occur during fermentation. Glass orplastic containers are therefore preferred. It has been foundparticularly convenient to culture the fungi in 55 gallon translucentplastic drums with just a cotton plug in the spigot. The insides of thedrums or other containers are first decontaminated, for example with adilute iodine solution, prior to use.

The preferred fungi useful in the invention are Basidiomycetes—i.e., aclass of fungi that coexist with, and depend for growth on, plants innature. Basidiomycetcs can be porous or gilled and preferred sources forthe spawns cultured in the method of the invention are the porous fungi,in particular those of the family Polyporaceae. The Polyporaceae cangenerally be classified as constituting genera that are brown rot fungior white rot fungi. The brown rot fungi degrade the white cellulose inwood on which they grow, thus leaving the brown lignin behind; the whiterot fungi do the opposite—they degrade the lignin and leave the whitecellulose behind. Thus, preferred fungi for use in the method of theinvention are brown rot Polyporus fungi, and in particular those of thegenera Bridgeoporus, Ceriporia, Daedalea, Laetiporus, Oligoporus, andPycnoporellus.

Thus, the invention can employ, in the specific culture conditionsrequired, various members of the Basidiomycete class, but preferablythose that are in the Polyporus family and in particular those that areof the brown rot type.

A particularly preferred fungus for use in the invention is Laetiporus,especially Laetiporus sulphureus. Laetiporus sulphureus “Sulphur shelf”or “Chicken of the Woods” is a wound parasite of hardwood trees. It iscommonly found in Hawaii on Eucalyptus robusta. Since this fungus livesin the heart wood of the tree, it is not noticeable on the outside ofthe tree. The fruiting body or mushroom appears as a sulphur or orangecolor bracket mushroom appearing every few years. The fungi feed on theheartwood and produce a cubical brown rot internally since the lignin isleft after the cellulose and hemicellulose have been dissolved byenzymatic action. Tree death occurs many years after infection hasstarted. The ability of this fungus to control the growth of the hosttree was not noticed in published literature nor has any plantstimulatory material been associated with this fungus. In controlledfungal liquid culture growth of the present invention, these plantgrowth factors have been produced in large quantities and have beendemonstrated to affect plant growth in plants outside the usual hostrange. As described herein, in order to utilize the plant stimulatorymaterial, the tissue degradation enzymes are destroyed by heating thefluid; the stimulatory material is heat stable at 100° C.

The fungal liquid culture of L. sulphureus has been shown to stimulateseed germination of corn, soybeans, lettuce, beans, grains and grasses;foliar spray has been shown to affect pineapples, coffee, tomato, taro,sugarcane and other crops. This fungus normally affects the growth of an100 ft tall eucalyptus tree weighing tons of biomass. The plantstimulatory effect is effective on smaller plants as well, with theproper dilution. The liquid culture fluid can be diluted 1:3,000, forexample, with water and still be effective. In nature, Laetiporus fungihave balanced the various plant growth regulators production in order tostimulate a large host tree with massive girth and root system. Thisbalance is applied on smaller plants and trees with desirable effects.

The fungal spawn is thus comprised preferably of a Basidiomycetc, morepreferably of a brown rot Basidiomycetc, and more preferably aLaetiporus fungus, grown under suitable conditions for the particularchoice of fungal organism. The nutrient medium for the fungus containssuitable components that are specifically tailored to the fungusemployed, but must always contain, of course, a source of carbon, asource of nitrogen, and relevant vitamins and cofactors. The spawn isproduced by culturing for a suitable time period sufficient to providesufficient fungal inoculum so that a mycelial mat will be formed in theculture medium of the invention. Typical time for formation of the spawnfrom an initial inoculation range from 5 days-100 days.

A commercially available liquid fungal spawn useful in the invention maybe obtained from Kukui Spawn Co., formerly Maui Shiitake TradingCompany, both of Maui.

The liquid spawn is then used to inoculate the culture medium forpreparation of the LCF. The inoculated culture medium is culturedwithout agitation in the presence of light predominantly in the longwavelength portion of the visible spectrum, at a temperature of 15-37°C., preferably about 20° C. for a sufficient time to generate therequired levels of PGR. Typically, PGR are produced in useful quantitiesafter 30 days of culture, preferably after 45 days, and more preferablyafter 60 days.

Although agitation of the inoculated culture medium is to be avoided inorder to avoid disturbing the mycelial mat, aeration of the medium maybe desired. This can be supplied, for example, by bubbling oxygenthrough the medium, or other means whereby the mycelial mat is leftundisturbed. It has been found that sufficient oxygen is available evenwithout bubbling air through the medium and simply permitting aerationto occur through interaction with the mycelial surface.

By “long wavelength portion of the visible spectrum” is meant light witha wavelength of approximately 500-800 nm, preferably 600-750 nm. Otherwavelengths may be included, but the predominant wavelengths should bein the above range. Thus, as a percentage of total photons, the longwavelength portion should represent more than 50% of said photons.

PGR levels can be assessed using standard bioassay methods. Comparisonof soybean bean seedling growth at known concentrations of LCF used as asoil drench in potting soil are used as growth standards. The color ofthe LCF solution correlates to efficacy on soybean seedlings over time.Fertilizer rates have been set for both treated and control andseedlings are evaluated 14 days after application. Effects ofdifferences in total weight, root weight, root length, foliage weight,and height are measured for comparison with effects from Standard LCFsolutions. Alternatively, the color of the culture fluids can be used asan index as it has been found that a color change from yellow to deepwine red is correlated with the production of PGR.

The strength of a LCF solution is controlled by matching the color of aknown LCF solution “Standard” that has a useable dilution strength of1:500 on soybean seedlings. Usually a culture that has been incubatingfor 60 days will be diluted with water 1:2 to reach a color intensity ofthat will match the Standard. Longer incubation time leads to furtherdilution of the culture fluids to reach the Standard LCF levels.

As the culture matures, a mycelial mat will be grown, and the liquidportion of the culture can readily be removed aseptically whensufficient PGR production has occurred. Solids are removed from theharvested culture medium, preferably by filtration or alternatively bycentrifugation or other known means to separate out solids. The liquidportion is then subjected to heating to 100° C. and held for 10 minutes.This step denatures the enzymes such as cellulase, lipase andhemicellulases. Chemical/temperature protein extractions or membranefiltration could also be used. The denatured proteins can then be paperfiltered or removed by centrifugation. The liquid portion is thensubjected to suitable sterilization procedures, such as pasteurizationand ultrafiltration, preferably pasteurization. The resultingpasteurized “LCF” is then packaged in sterile containers. The cultureflask with the mycelial mat is then, if desired, refilled with sterile,cool nutrient solution for preparation of additional LCF. The first LCFproduction takes 60 days, the second requires only about 30 days sincethe mycelial mat has been established in the first run. The third andsubsequent production runs are 30 days long and may be continued untilthe culture vessel becomes contaminated.

By “culture filtrate” is meant the liquid portion of the culturedescribed. “Culture filtrate” is a commonly used term, despite the factthat recovery of this filtrate may not necessarily be effected by actualfiltration. Indeed, in many of the cultures of the present invention, amycelial mat is formed so that the culture filtrate may be removed bydecanting or by siphoning. “Culture filtrate” thus refers simply to theliquid portion of the culture.

“Sterilization” of the LCF of the invention can be effected by a varietyof means. Because pasteurization is the most practical, the compositionis referred to as LCF. However, other modes of sterilization could alsobe effected, such as ultrafiltration or inclusion of antibiotics orsanitizers such as Idophor.

In addition to the sterilized culture filtrate, the LCF compositions ofthe invention also include the material retained upon filtration of theculture which can be dried. This material also contains PGR and can beused in a manner similar to the sterilized filtrate.

The mycelial mat can be reused after-removal of the medium forharvesting the LCF. Typically, the medium can be removed through tubingfrom under the mat and replaced by new sterile medium. As typically themat is broken during removal of the prior medium, the new sterile mediumcan simply re-poured into the container and the portions of the matre-assemble and continue to grow.

In addition to the production of plant growth regulators according tothe methods of the invention, it has been found that the mediumcontains, as well, elicitors of materials which constitute the defensesof plants against pathogens. These defense mechanisms, known generallyas phytoalexins are engendered in plants which are infected withparasites or pathogens. A general discussion of these mechanisms isfound in the report of a lecture entitled “How Plants Defend ThemselvesAgainst Pathogens,” Lecture 8 from U. of Idaho Plant Science 405/504courses found at www.uidaho.edu/ag/plantdisease. As outlined, ingeneral, structural defenses involve, for example, formation of waxes,cork layers, abscission layers, and the like which constitute barriersfor disruption of plant metabolism. Metabolic defenses includepreexisting defenses as well as those elicited by the infection withpathogens. These defenses include production of the toxic substancesdesignated as phytoalexins. The LCF of the invention is able to elicitresponses of this type.

Thus, one aspect of the invention combines the plant growth regulationeffects of the LCF with eliciting production of phytoalexins. Thisaspect can be strengthened by addition of known phytoalexin inhibitors,such as the product called Messenger containing the protein Harpin,manufactured by Eden Bioscience.

The LCF is diluted to a suitable concentration for application to cropsor trees. Dilutions of 1:100-1:2,000 or 1:5,000 can be used, dependingon the concentration of PGR and the desired effects. The level ofdilution depends, of course, on the initial concentration of the PGR,,the manner in which the material is to be applied, and a number of otherfactors that are well within ordinary skill to determine. LCF dilutionsas high as 1:6,000 or even greater degrees of dilutions 1:10,000, havebeen found effective in many cases. The LCF may also be dried on aninert support such as talc or diatomaceous earth for application, or maybe dried onto a granular fertilizer to boost the performance of thefertilizer. In general, the diluted LCF may be applied in anyconventional manner, such as including in a planting dip or foliarspray, adding through drip irrigation systems, mixing with potting soil,applying to surrounding soil for seedlings, etc.

In one embodiment application rates are 6-8 oz of LCF per 30 gal ofwater per acre when used as a spray or soil supplement. However,applications of larger amounts of a liquid are preferred. Preferably,the total liquid applied to an acre will be 100-500 gallons with thedilution approximating 1 pint-1.5 quarts diluted to 100-200 gallonswater. Thus typical applications would use 1 quart of LCF as sterilizedmedium in 125-200 gallons of water per acre applied as a spray or 2quarts of the sterilized culture filtrate LCF diluted in 325 gallons ofwater per acre.

However, as stated above, the culture filtrate LCF may also be driedunto a granular substance and applied as a dried material.

The LCF and its dilutions or other formulations can also be mixed withother acidic materials such as pesticides, other nutrients and/orfertilizers for combined application. Mixing with basic fertilizers orsolutions should be avoided. A particularly preferred mixture is thatwith the surfactant blend crop adjuvants described in PCT publication WO96/38590, which is incorporated herein by reference. Alternatively,nematicide mixtures which consist entirely of exempt ingredients, suchas mixtures of sodium lauryl sulfate, molasses, safflower oil and cheesecould also be used. These are described in copending application Ser.No. 60/390,289, filed 21 Jun. 2002 and incorporated herein by reference.As stated above, also included in the formulation may be elicitors ofthe phytoalexin defense proteins or metabolites.

By way of illustration, the LCF can be supplied as a 1% (wt/wt) coatingon fertilizer pellets designed for turf grass or for vegetables andtrees. A 2% (wt/wt) coating on diatomaceous earth is a suitable seedtreatment flour for small seeds such as lettuce, tomato, cabbage andeggplants; higher percentages of LCF coated on diatomaceous earth areuseful for larger seeds. For example, a 6.5% coating is suitable forcorn, beans, soybeans, peas and cucumbers. Bare root seedlings, corms,pineapple crowns, and other vegetative planting material can be dustedwith this flour to enhance growth.

A convenient mode of application is to add about ½ teaspoon of the flourto an ounce of seed in a plastic bag. After closing the bag, the bag isshaken to coat the seeds with flour, the excess flour is recovered andthe resulting seeds have a fine coat of the flour.

In general, the LCF should be stored at room temperature, out of directsunlight, under dry conditions. While the LCF is environmentally safe,it should not be ingested and should not be allowed to remain on theskin for extended periods.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Preparation of Pasteurized LCF

Medium was prepared from:

-   -   20 gal of high-fiber pineapple juice retentate obtained from        ultrafiltration,    -   1 gal of pineapple syrup,    -   20 gal of molasses water (5 gal of molasses mixed with 40 gals        of water), and    -   5 gal of soft ripe-stage papaya puree, optionally including        skin, seeds and pulp.        The total volume was then adjusted to 50 gal with additional        retentate and/or molasses water. The components were mixed in an        open plastic drum and transferred to stainless steel pots,        brought to a boil, and held at 100° C. for at least 30 min. The        hot slurry was transferred to a sanitized, white translucent, 55        gal plastic bioprocessing drum and cooled to room temperature.        Cooling takes several days.

The cooled medium was inoculated with fungal starter culture (see below)under a laminar flow-hood and one or two sterilized cotton plugs wereinstalled in the 55 gal drum. The drum was then incubated in anair-conditioned, lighted room for 60 days undisturbed. The lighting wassupplied by Agrolights or deluxe warm white light. Cool white light isnot satisfactory.

After 60 days, the liquid portion of the culture was removed andfiltered to remove solids. The filtered liquid was heated to 100° C. for10 minutes to denature soluble proteins. The heated medium was thenfiltered and reheated to 100° C. for 30 minutes to pasteurize. Thepasteurized product was filled into sanitized bottles and kept at roomtemperature for storage. The pH of the LCF prepared in this Example is2.5.

The fungal starter culture used in this Example was obtained from KukuiSpawn Co., formerly Maui Shiitake Trading Company, Hawaii. The entire 1liter container of liquid spawn is added aseptically to thebioprocessing drum of cooled nutrient solution. Usually two bottles ofspawn liquid culture is added per 50 gallon bioprocessing drum.

EXAMPLE 2 Application to Plants

One ounce of LCF as prepared in Example 1 was diluted into 5 gal ofwater. This mixture was applied to soil around coffee trees.

One ounce of LCF-was diluted into 5 gal of water and used to treat cornplants, which grew 60% more bulk than control plants. Improved resultswere shown with dilution of 1 oz LCF to 10 gal of water.

A similar dilution of 1 oz LCF to 5 gal of water was used to spray Manoalettuce which yielded 50% more leaf weight than controls.

Cucumber seeds were treated with 6.5% LCF flour prior to planting andthe plants grew 39% heavier than controls.

One ounce of LCF diluted to 5 gal of water was applied to pineapplecrowns and resulted in faster growing pineapples; improved growthresults were also observed when older plants were foliarly sprayed withthis dilution or when 3-year-old pineapple plants were dipped in thisdilution.

A dilution of 1 oz LCF in 1 gal of water was used as a corm/planting dipfor dry land taro, sweet potatoes and yams.

Taro seedlings grew 160% heavier than control plants in six weeks as aresult.

In application to sugar cane, the dilution required varied with varietyof plant. In some instances, 1 oz of LCF and 2½ gal of water wassuccessful in obtaining 50% increase in shoot diameter.

Turf grass was made to grow at a faster rate by spraying with 1 oz LCFdiluted in 5 gal of water.

LCF has also been used as a soil drench of 29 mls of a 1:500 dilutionper seedling pot to treat Douglas fir, resulting in 10%-20% growthwithin four weeks.

LCF can be mixed with polymers for a dry seed coating treatments ofgrains, vegetables and other crops. Percentage LCF will vary with thecrop.

LCF can be used to alleviate nematode infestation symptoms. A 1:500dilution of 5 gallons per infested coffee tree. Three applications in 5months will increase roots, foliage branches, increase fruit uniformityand increase production.

LCF may be used to reduce plant disease incidences such as fungal leafspots and bacterial rot of plants. Onion Bulb Rot may be reduced with a1:1600 dilution of LCF applied foliarly every three weeks.

A weak solution of LCF, 0.1% solution may be used to extend the life ofcut flowers, foliage and Christmas trees.

LCF may be used to reduce frost, insect and chemical damage to plantswith a 1:500 foliar or soil drench application.

Pineapple shoots were tested by dipping shoots in a solution containing800, 1000, or 2000 parts per million of the LCF sterilized culturefiltrate for 10 minutes prior to planting in potting soil with granularfertilizer. The shoots were also dipped in a microemulsion surfactantblend nematicide at the same time. While the controls had an averageroot weight of 6.9 grams and an average root length of 29.4 inches, the800 part per million LCF treated plants had root weights of 8.2 gramsand root lengths of 33.35 inches; those treated with 1000 parts permillion LCF showed root weights of 7.4 grams and root lengths of 41.4inches, a 41% increase over control. However at 2000 parts per million,toxic effects appeared and the root weights and lengths were less thancontrols.

Garbanzo beans were soaked in a dilution of the sterilized culturefiltrate LCF diluted 1 ounce in ½ gallon or 1 gallon of water. The beanswere treated at a ratio of 1 ml of the diluted material per 100 grams ofbeans. The beans were then dried and planted and showed an increase of22% in the yield of beans harvested as compared to untreated controls.

In other applications, the LCF composition treatments of the inventionwere combined with treatments with flower stimulators, herbicides, andturf grass growth inhibitors.

An increased fruit weight of 56% was obtained when LCF was applied totomatoes.

1. A composition comprising plant growth regulating substances whichcomposition is a sterilized culture filtrate recovered from a culture ofthe spawn of a Basidiomycete fungus incubated in a medium containing atleast 10% available carbohydrate in the presence of long wavelengthlight, and in the substantial absence of agitation, wherein said mediumhas a BRIX value of 12-15 and further contains potassium ion andcarotene, and wherein said filtrate is the liquid portion of saidculture from which proteins have been denatured and removed.
 2. Thecomposition of claim 1, wherein said medium comprises molasses and/orpineapple or papaya syrup or juice.
 3. The composition of claim 1,wherein said medium contains sufficient carotene to impart a yellowcolor, and K⁺of 0.01%-0.1% wt/vol.
 4. The composition of claim 1,wherein the Basidiomycete is a Polyporus fungus.
 5. The composition ofclaim 4, wherein the Polyporus is a brown rot Polyporus.
 6. Thecomposition of claim 5, wherein the brown rot Polyporus is a Laetiporus.7. A formulation for application to enhance plant growth and/ordevelopment which formulation comprises an effective amount of acomposition comprising plant growth regulating substances, whichcomposition is a sterilized culture filtrate recovered from a culture ofthe spawn of a Basidiomycete fungus incubated in a medium containing atleast 10% available carbohydrate in the presence of long wavelengthlight and in the substantial absence of agitation, wherein said mediumhas a BRIX value of 12-15 and further contains potassium ion andcarotene , wherein said filtrate is the liquid portion of said culturefrom which proteins have been denatured and removed and wherein saidfiltrate is optionally dried.
 8. The formulation of claim 7 whichcomprises diatomaceous earth or fertilizer particles coated with saidcomposition.
 9. A method for preparing a composition comprising plantgrowth regulators, which method comprises sterilizing a filtrate of aculture of the spawn of a Basidiomycete fungus, whereby the culture hasbeen grown in medium containing at least 10% available carbohydrate inthe presence of long wavelength light and in the substantial absence ofagitation, wherein said medium further contains potassium ion andcarotene, and wherein proteins have been denatured and removed from thefiltrate.
 10. The method of claim 9, wherein said sterilizing is bypasteurization.
 11. A method for preparing a composition containingplant growth regulators, which method comprises culturing the spawn of aBasidiomycete fungus in a medium containing at least 10% availablecarbohydrate in the presence of long wavelength light and in thesubstantial absence of agitation, wherein said medium further containspotassium ion and carotene; recovering a culture filtrate; denaturingsoluble proteins in the filtrate and removing said denatured proteins;and sterilizing the remaining culture filtrate to obtain saidcomposition.
 12. The method of claim 11, wherein the medium containssufficient carotene to impart a yellow color, and K⁺of 0.01%-0.1%wt/vol.
 13. The method of claim 11, which said culture filtrate isrecovered by filtering said culture.
 14. The method of claim 11, whereinsaid denatured proteins are removed by filtering.
 15. The method ofclaim 11, wherein said sterilizing is by pasteurization.
 16. The methodof claim 11, wherein the Basidiomycete is a Polyporus fungus.
 17. Themethod of claim 16, wherein the Polyporus is a brown rot Polyporus. 18.The method of claim 17, wherein the brown rot Polyporus is a Laetiporus.19. A method to enhance plant growth, development or bulk, which methodcomprises contacting the seeds or at least a portion of said plant withthe formulation of claim
 7. 20. The method of claim 19, wherein saidformulation further contains at least one pesticide and/or at least onenutrient and/or at least one herbicide.
 21. The method of claim 19,wherein said formulation further contains at least one elicitor ofphytoalexin production.
 22. The method of claim 19 which furthercomprises contacting the seeds or at least a portion of said plant withat least one pesticide and/or at least one nutrient and/or at least oneherbicide.